梓醇对大鼠骨髓间充质干细胞体外增殖和成软骨分化能力的影响

Effect of Catalpol on Proliferation and Chondrogenic Differentiation of Rats Bone Mesenchymal Stem Cells in Vitro

目的探讨梓醇对大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)体外增殖和成软骨分化能力的影响。方法全骨髓贴壁筛选法培养BMSCs,取第3代细胞用于实验,并作成骨、成脂分化鉴定。CCK-8法检测25、50、100、200μmol/L梓醇在12、24、36 h对BMSCs增殖效应,选取含50、100、150μmol/L梓醇的经典诱导液、经典诱导液、普通培养基诱导BMSCs成软骨分化,诱导3周后,HE染色观察成软骨大小。选取相同浓度的梓醇、经典诱导液、普通培养基培养BMSCs12 h后,实时荧光定量PCR检测COL1、Aggrecan和SOX9基因表达。结果不同浓度的梓醇对BMSCs均无毒性作用,24 h后不同浓度的梓醇都可促进BMSCs增殖,随着梓醇浓度增大及作用时间的延长,BMSCs增殖也明显增加,与空白正常组比较均有统计学意义(P<0.01)。HE染色观察,加入含150μmol/L梓醇的经典诱导组诱导的软骨小球较单纯经典诱导液组大,而PCR结果显示与空白组比较,不同浓度组梓醇均能提高COL1、Aggrecan和SOX9基因表达(P<0.01),与经典诱导组比较,低、中剂量的梓醇组可以提高COL1基因的表达(P<0.05),高剂量梓醇可提高Aggrecan基因的表达(P<0.05),高、中、低剂量梓醇组都可以提高SOX9的基因的表达(P<0.05)。然而,经典诱导组SOX9表达量与空白组比较没有明显统计学差异(P>0.05)。结论梓醇能促进大鼠BMSCs体外增殖,与时间和浓度成正相关,亦能促进大鼠BMSCs体外成软骨分化,可能与促进COL1、Aggrecan基因表达相关。

Objective To discuss the effect of catalpol on proliferation and chondrogenic differentiation of rats bone mesenchymal stem cells(BMSCs)in vitro.Methods BMSCs were isolated from whole rat bone marrow and amplified by adherent culture methods and then BMSCs at passage 3 were used and identified by osteogenic differentiation and adipogenic differenctiation.CCK-8 was employed to detect proliferation of BMSCs treated with catalpol at concentrations of 25,50,100,200μmol/L after 12 h,24 h,36 h respectively.BMSCs were induced chondrogenic differentiation with classic induction liquid containing catalpol at concentrations of 50,100,150μmol/L,classic induction liquid and ordinary medium.After 3 weeks,the size of cartilage stained with HE was observed.BMSCs were treated with catalpol at the same concentrations,classic induction liquid and ordinary medium after 12 h and then we detected expressions of COL1,Aggrecan and SOX9 mRNA by real time fluorescent quantitative PCR.Results Catalpol at different concentrations were non-toxic intervening BMSCs and promoted BMSCs proliferation after 24 h.Following by concentrations of catalpol increasing and acting time,BMSCs proliferation was markedly increased with statistic difference compared with that of the blank control group(P<0.01).The size of cartilage in classic induction liquid containing catalpol at concentration of 150μmol/L was larger than that in classic induction liquid group by HE stainting.PCR showed that compared with blank control group,catalpol at different concentrations promoted the expressions of COL1,Aggrecan and SOX9 mRNA in BMSCs(P<0.01).Compaired with classic induction liquid group,the low and medium dose of catalpol promoted the expression of COL1 mRNA(P<0.05),the high dose of catalpol promoted the expression of Aggrecan mRNA(P<0.05)and the low,medium and high dose of catalpol promoted the expression of SOX9 mRNA(P<0.05).While there was no statistic difference in the expression of SOX9 mRNA between the blank control group and the classic induction liquid group(P>0.05).Conclusions Catalpol could promote BMSCs proliferation in vitro,correlated with time and concentration positively.And it also could promote BMSCs chondrogenic differentiation in vitro and may be related to accelerating the expressions of COL1 and SOX9 mRNA in BMSCs.